The nuclear regulatory proteins required for erythroid development are not entirely defined. In other developmental systems proteins of the basic helix-loop-helix (bHLH) family are often involved in specifying cell fate. The bHLH factor known as SCL (stem cell leukemia) (also known as tal- 1/TCL5) is a potential candidate for an important role in erythroid development. In this project the murine SCL gene will be inactivated by gene targeting in embryonic stem cells, and mice homozygous for deficiency of SCL will be generated. The phenotype of these mice will be characterized throughout embryogenesis and adult life (if viable). In addition, ES cells homozygous for SCL deficiency will be generated from single knock-out cells in order to assess hematopoietic and erythroid development in vitro. If the phenotypes in vitro or in vivo are not striking in the absence of SCL, a systematic search for potentially redundant proteins will be undertaken, and involve cross-breeding of SCL heterozygotes with other bHLH knock-outs (e.g. Lyl). To further investigate the role of SCL in blood cell development, a zebrafish SCL cDNA clone will be isolated in collaboration with Core B, which will be used to examine the pattern of SCL RNA expression in normal zebrafish development, and in abnormal hematopoietic development exhibited by the bloodless mutant and other zebrafish mutants to be provided from investigators worldwide. Through combined study of SCL in mice and zebrafish the role of this protein in erythroid development should be established.